The present invention is directed towards specific knockout animals and their use as animal models. More specifically, the knockout animals contain a disruption in the genes encoding p19INK4d and p27KIP1. Corresponding cells which are amenable to tissue culture are also part of the invention, as are methods of using such cells, including their use as a potential source of differentiated neuronal cells.
The cell cycle for growing cells can be divided into two periods: (1) the cell division period, when the cell divides and separates, with each daughter cell receiving identical copies of the DNA; and (2) the period of growth, known as the interphase period. For the cell cycle of eucaryotes, the cell division period is labeled the M (mitotic) period. The interphase period in eucaryotes is further divided into three successive phases: G1 (gap 1) phase, which directly follows the M period; S (DNA synthetic) phase, which follows G1; and G2 (gap 2) phase, which follows the S phase, and immediately precedes the M period. During the two gap phases no net change in DNA occurs, though damaged DNA may be repaired. On the other hand, throughout the interphase period there is continued cellular growth and continued synthesis of other cellular components. Towards the end of the G1 phase, the cell passes a restrictive (R) point and becomes committed to duplicate its DNA. At this point, the cell is also committed to divide. During the S phase, the cell replicates DNA. The net result is that during the G2 phase, the cell contains two copies of all of the DNA present in the G1 phase. During the subsequent M period, the cells divide with each daughter cell receiving identical copies of the DNA. Each daughter cell starts the next round of the growth cycle by entering the G1 phase.
The G1 phase represents the interval in which cells respond maximally to extracellular signals, including mitogens, anti-proliferative factors, matrix adhesive substances, and intercellular contacts. Passage through the R point late in G1 phase defines the time at which cells lose their dependency on mitogenic growth factors for their subsequent passage through the cycle and, conversely, become insensitive to anti-proliferative signals induced by compounds such as transforming growth factor, cyclic AMP analogs, and rapamycin. Once past the R point, cells become committed to duplicating their DNA and to undergoing mitosis, as noted above, and the programs governing these processes are largely cell autonomous.
In mammalian cells, a molecular event that temporally coincides with passage through the R point is the phosphorylation of the retinoblastoma protein (Rb). In its hypophosphorylated state, Rb prevents the cell from exiting the G1 phase by combining with transcription factors such as E2F to actively repress transcription from promoters containing E2F binding sites. However, hyperphosphorylation of Rb late in G1 phase prevents its interaction with E2F, thus allowing E2F to activate transcription of the same target genes. As many E2F-regulated genes encode proteins that are essential for DNA synthesis, Rb phosphorylation at the R point helps convert cells to a pre-replicative state that anticipates the actual G1/S transition by several hours.
Regulation of the human cell cycle requires the periodic formation, activation, and inactivation of protein kinase complexes that consist of a regulatory xe2x80x9ccyclinxe2x80x9d subunit and a catalytic xe2x80x9ccyclin dependent kinasexe2x80x9d (CDK) subunit. Cell cycle-dependent fluctuations in the levels of many of the cyclin proteins contribute to the activation of these protein kinase complexes. For example, cyclin B participates in the regulation of the G2/M transition by its association with its catalytic subunit, p34cdc2, whereas cyclin A, in complexes with both p34cdc2 and CDK2, is essential for the completion of S-phase and entry into G2-phase. Complexes formed between the D-type cyclins and either CDK4 or CDK6 integrate growth factor signals and the cell cycle, allowing cells to progress through G1-phase. This particular cell cycle pathway is specifically altered during tumorigenesis, presumably due to its role in responses to mitogenic stimulation. Alterations have been identified in many components of this pathway, including the D-type cyclins, CDKs, and cyclin dependent kinase inhibitors (CKIs). Another G1-phase cyclin, cyclin E, in conjunction with its catalytic subunit CDK2, appears to be essential for progression from G1-phase into S-phase and the initiation of DNA replication. Cyclin E and CDK2 do not appear to be directly targeted during tumorigenesis, quite possibly due to their essential nature. [See generally, Sherr, Cell 73:1059-1065 (1993); and Sherr, Cell 79:551-555 (1994)].
A class of novel polypeptides that are collectively known as CDK inhibitors (CKIs) can negatively regulate cyclin/CDK activity by associating with these complexes. These so-called xe2x80x9ccell cycle brakesxe2x80x9d act to inhibit cyclin/CDK complexes by binding specifically to either CDK, (i.e.,the INK4s, see below) or the cyclin/CDK complexes (i.e., KIP1/CIP1, see below). CKI activity and levels are cell cycle regulated allowing these proteins to function as inhibitors of their cognate cyclin/CDK complexes for very limited periods during the cell cycle.
There are two types of CKIs that have been identified, the INK4s, and the CIP/KIPs [Sherr and Roberts, Genes Devel. 9:1149-1163 (1995)]. The INK4 family of inhibitors comprises four members, p16INK4a, p15INK4b, p18INK4c, and p19INK4d, which specifically bind to and inhibit G1-specific CDK4 and CDK6, and thereby prevent phosphorylation of the retinoblastoma (Rb) protein and S phase entry. The CIP/KIP family of inhibitors includes p21CIP1, p27KIP1, and p57KIP2, and unlike the INK4 proteins, can inhibit all cyclin/CDK complexes [Harper and Elledge, Curr. Opin. Genet. Dev. 6:56-64 (1996)]. This apparent redundancy in CDK complex inhibitors has been explained as a method for organisms to govern transitions through the R point in different cell types responding to a plethora of distinct extracellular signals.
Despite their apparent biochemical redundancy, the CKIs are differentially expressed during mammalian (e.g., mouse) development and in adult tissues, showing some overlapping expression patterns. Deletion of a single CKI or in combination with another in the mouse leads to specific phenotypes, but in spite of their reported expression in the central nervous system (CNS) none give rise to a reported phenotype in the brain. For example, mice lacking p57KIP2, die soon after birth and display developmental defects mimicking those observed in human patients with Beckwith-Weideman syndrome [Zhang et al., Nature 387:151-158 (1997); Yan et al., Genes and Development 11: 973-983 (1997)]. Mice lacking p27KIP2 develop organomegaly, neurological conditions i.e., display retinal dysplasia, female sterility, and benign pituitary adenomas [Fero et al., Cell 85:733-744 (1996); Kiyokawa et al., Cell 85:721-732 (1996); Nakayama et al., Cell 85:707-720 (1996)]. However, mice deleted for the other CKIs examined heretofore, do not display apparent phenotypes. Mice lacking p21CIP1 [Deng et al., Cell 82:675-684 (1995)] are developmentally normal and do not develop spontaneous tumors. Mice lacking p18INK4c develop gigantism, widespread organomegaly and pituitary adenomas by 10 months of age [Franklin et al., Genes and Development 12:2899-2911 (1998)], whereas mice lacking both p18INK4c and p27KIP1 develop pituitary adenocarcinomas with an accelerated onset from what is seen in the p18INK4c-double null animals (i.e., animals lacking both p18INK4c alleles) [Franklin et al., Genes and Development 12:2899-2911 (1998)]. Similarly, deletion of p27KIP1 and p57KIP2 leads to aberrant proliferation of these cells due to inhibition of cell cycle exit and differentiation in these tissues. In wild type mice, both p27KIP1 and p57KIP2 are expressed in the lens fiber cells and in placental trophoblasts, [Zhang et al., Genes and Development 12:3162-3167 (1998)].
Development of the central nervous system (CNS) requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. However, despite the obvious need for replacement of neuronal cells in cases of neural injuries and diseases, to date, no such cell source is available. Therefore, there is presently a need for methodology that can provide an avenue for stimulating the growth of neuronal populations. Furthermore, there are relatively few if any animal models for diseases that include symptoms such as bradykinesia. Therefore, there is a need for animal models for this and other neurological maladies and disorders. In addition, like neurons of the CNS, regeneration of sensory cells of the inner ear does not occur following their loss. Methodology for stimulating the proliferation of these cells or their precursors the non-sensory supporting cells of the auditory and vestibular epithelium is needed to treat hearing and balance disorders caused by a loss or malfunction of these cells.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
The present invention provides non-human animals that have been manipulated to be missing all or essentially all of an activity of one or more specific gene/allele product(s). In a preferred embodiment of this type the non-human animal has been manipulated so as not to express functional p19INK4d and p27KIP1 proteins. Preferably the non-human animal is a mammal. In addition, the present invention provides cells, preferably animal cells, and more preferably mammalian cells that have been manipulated to be missing all or essentially all of an activity of the p19INK4d protein, and to be missing all or essentially all of an activity of the p27KIP1 protein. In a particular embodiment the mammalian cell is a human cell.
The present invention also provides methods of making and using these cells and non-human animals. The present invention further provides methodology for stimulating growth of cells that do not generally proliferate, such as neuronal populations or sensory cells of the inner ear. The neuronal populations can be employed to replenish those lost due to degenerative diseases such as Alzheimer""s disease or Parkinson""s disease, or alternatively lost through catastrophic insults such as during strokes or traumatic injury such as spinal chord injuries. Stimulating the growth of cells in the inner ear that do not otherwise proliferate can be employed to replenish the loss of sensory hair cells lost due to chemotherapy, disease or other ototoxic shock.
In still another aspect of the present invention the cells and non-human animals of the present invention can be used to identify potential modulators of cell growth. In a particular embodiment, modulators of neuronal cell growth are identified. In another embodiment, modulators of the growth of sensory hair cells of the inner ear are identified. All of these modulators are also part of the present invention. Neuronal growth modulators that stimulate neuronal cell growth, for example can also be used to treat degenerative diseases such as Alzheimer""s disease or Parkinson""s disease, or alternatively treat the loss of neuronal cells due to the result of a catastrophic insult such as that which occurs during a stroke or a traumatic injury such as a spinal chord injury. Modulators that stimulate sensory hair cell growth can be used to treat hearing and balance disorders caused by a loss or malfunction of these cells.
The knockout animals and/or corresponding cells of the present invention can be manipulated to be incapable of expressing a functional protein from one or more specified alleles and/or genes by any means known in the art. For example, a knockout animal and/or corresponding cell can be manipulated to comprise a disruption in an endogenous allele(s) and/or gene(s) [e.g., one encoding the p19INK4d protein] thereby preventing the expression of the corresponding functional protein. Alternatively, a knockout animal and/or corresponding cell can be manipulated to comprise a dominant mutant allele. In a particular embodiment of this type nucleic acid(s) encoding one or more CDKs and/or cyclins are constructed to be under the control of a neuron-specific promoter (e.g., the enolase promoter) and are thereby overexpressed in the neuronal cell, and bind all of the free p19INK4d protein and/or p27KIP1 protein. In an alternative embodiment, nucleic acid(s) encoding one or more CDKs and/or cyclins are constructed to be under the control of a sensory or supporting cell specific promoter such as the Math-1 promoter/enhancer or S-100 promoter.
In yet another embodiment, a knockout animal and/or corresponding cell can be treated with one or more antisense nucleic acids for one or more specific gene(s) thereby inhibiting the expression of the specific gene(s). In still another embodiment the gene(s) encoding the specific functional protein(s) can be constructed such that the expression of the protein(s) is under the control of an inducible promoter, and this expression is conditionally repressed. In still another embodiment the cell and/or non-human animal is treated with/administered inhibitory compound(s) that prevent the expression and/or activity of the p19INK4d protein and/or p27KIP1 protein.
Therefore, one aspect of the present invention includes a non-human transgenic knockout animal that comprises a homozygous disruption in its endogenous p19INK4d gene and a heterozygous disruption in its endogenous p27KIP1 gene, such that the homozygous disruption prevents the expression of a functional p19INK4d protein from either endogenous p19INK4d allele and the heterozygous disruption in the endogenous p27KIP1 gene prevents the expression of a functional p27KIP1 protein from at least one endogenous p27KIP1 allele. The male knockout animal of this type relative to the animal with a wild type p19INK4d gene and p27KIP1 gene manifests at least about a 20% to 40% reduction in testicular size. The female knockout animal of this type when mated with the corresponding male knockout animal has the phenotype of bearing male progeny that manifest at least about a 20% to 40% reduction in testicular size relative to the corresponding male having a wild type p19INK4d gene and p27KIP1 gene.
The present invention further provides an isolated cell from such a non-human knockout animal. In a preferred embodiment the non-human transgenic knockout animal is a mammal. In a particular embodiment of this type, the non-human mammalian transgenic knockout animal is a knockout mouse.
The present invention further provides a non-human transgenic knockout animal that has a homozygous disruption in its endogenous p19INK4d gene such that the expression of a functional p19INK4d protein is prevented, and a homozygous disruption in its endogenous p27KIP1 gene such that the expression of a functional p27KIP1 protein is prevented. In a preferred embodiment of this type, the non-human transgenic knockout animal is a mammal. In a particular embodiment of this type, the non-human mammalian transgenic knockout animal is a knockout mouse.
In one embodiment a knockout animal that has a homozygous disruption in its endogenous p19INK4d gene and a homozygous disruption in its endogenous p27KIP1 gene develops one or both of the following characteristics during its lifetime: (i) bradykinesia, and (ii) proprioceptive abnormalities In a particular embodiment of this type the knockout animal exhibits one of these characteristics. In a more preferred embodiment of this type knockout the animal exhibits both of these characteristics. In a particular embodiment of this type, the knockout animal is a knockout mouse that has a life expectancy of between about 14 to about 24 days after birth.
The present invention further provides an isolated cell from a non-human knockout animal that has a homozygous disruption in its endogenous p19INK4d gene and a homozygous disruption in its endogenous p27KIP1 gene. In a particular embodiment of this type, the cell is a neuronal cell that is a member of a subpopulation of neurons. In one such embodiment the cell subpopulation is a normally dormant neuron of the hippocampus, cortex, pons, or hypothalamus. In a preferred embodiment of this type such a cell can continue to proliferate after migrating to its final positions in the brain.
In still another embodiment, the present invention provides a knockout animal that has a homozygous disruption in its endogenous p19INK4d gene such that the expression of a functional p19INK4d protein is prevented, a homozygous disruption in its endogenous p27KIP1 gene such that the expression of a functional p27KIP1 protein is prevented and further comprises a homozygous disruption in its endogenous p57KIP2 gene such that the expression of a functional p57KIP2 proteins also prevented.
Another aspect of the present invention is a cell that has been manipulated to be incapable of expressing a functional p19INK4d protein and has been further manipulated to be incapable of expressing a functional p27KIP1 protein from at least one endogenous p27KIP1 allele. The cell is preferably a mammalian cell. In a still another embodiment, the cell has been manipulated such that it cannot express a functional p27KIP1 protein and is incapable of expressing a functional p19INK4d protein from at least one endogenous p19INK4d allele. In a preferred embodiment the cell has been manipulated such that it can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein. In still another embodiment, a cell that has been manipulated such that it can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein, has been further manipulated to be incapable of expressing a functional p57KIP2 protein from at least one endogenous p57KIP2 allele. In a preferred embodiment of this type the cell has been further manipulated to be incapable of expressing a functional p19INK4d protein, a functional p27KIP1 protein, and a functional p57KIP2 protein.
The mammalian cells of the present invention can be of any cell type including a neural stem cell, a neuronal cell, a glial cell, a hemopoietic stem cell, a B lymphocyte, a T lymphocyte, a natural killer cell, a dendritic cell, a macrophage, a megakaryocyte, a keratinocyte, and a retinal cell. In a preferred embodiment, the mammalian cell is a neuronal cell. In another embodiment, the mammalian cell is a sensory hair cell or non-sensory supporting cell of the inner ear epithelium including the organ of Corti and/or the vestibular system, and/or a stem cell of the auditory or vestibular sensory epithelium. In a preferred embodiment, this type of cell can continue to proliferate after migrating to its final position in the organ of Corti.
The present invention further provides a method for selecting a potential therapeutic agent for use in the treatment of a motor condition/disorder (e.g., one in which bradykinesia is a symptom) and/or proprioceptive, hearing or balance abnormalities. One such embodiment comprises administering a potential therapeutic agent to a non-human knockout animal that can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein, and measuring the response of the knockout animal to the potential therapeutic agent. The response of the knockout animal is then compared to that of an animal that expresses both functional p19INK4d protein and functional p27KIP1 protein. A potential therapeutic agent is selected based on the difference in response observed between the knockout animal in the presence and absence of the potential therapeutic agent relative to that of the animal expressing both functional p19INK4d protein and functional p27KIP1 protein. In a preferred embodiment the non-human transgenic knockout animal is a mammal. In a particular embodiment of this type, the non-human mammalian transgenic knockout animal is a knockout mouse.
The present invention further provides a method of proliferating neuronal cells or sensory and nonsensory cells of the inner ear comprising culturing a neuronal cell, or a sensory or nonsensory cell of the inner ear that can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein. The cells that have been proliferated and/or generated by this method are also part of the present invention. In a particular embodiment of this type, the cell is obtained from a knockout animal that can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein. In a preferred embodiment of this type, the cell is obtained from a knockout animal that comprises a homozygous disruption in its endogenous p19INK4d gene such that the expression of a functional p19INK4d protein is prevented, and a homozygous disruption in its endogenous p27KIP1 gene such that the expression of a functional p27KIP1 protein is prevented.
The present invention also includes a method of proliferating a mammalian cell such as a neuronal cell, or a sensory or nonsensory cell of the inner ear comprising treating the cell with an inhibitor of the expression of the p19INK4d gene and an inhibitor of the expression of the p27KIP1 gene. In another embodiment the method of proliferating a mammalian cell such as a neuronal cell, or a sensory or nonsensory cell of the inner ear comprises treating the cell with an inhibitor of the p19INK4d protein and an inhibitor of the p27KIP1 protein. In still another embodiment the method of proliferating a mammalian cell such as a neuronal cell, or a sensory or nonsensory cell of the inner ear comprises treating the cell with an inhibitor of the expression of the p19INK4d gene and/or an inhibitor of the p19INK4d protein, and an inhibitor of the expression of the p27KIP1 gene and/or an inhibitor of the p27KIP1 protein.
The present invention also provides a method of treating a subject having a hearing or balance disorder. The present invention further provides a method of treating a subject having a neurological degenerative disease such as Alzheimer""s disease or Parkinson""s disease, or one that has suffered a traumatic injury in which neuronal cells are damaged comprising implanting into the subject a neuronal cell of the present invention, which has been proliferated and/or generated by a method of the present invention into the brain of the subject. In a particular embodiment the neuronal cell is implanted into the brain of the subject. In a preferred embodiment the neuronal cell can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein.
In another embodiment, the mammalian cell, such as a neuronal cell or a sensory or nonsensory cell of the inner ear, has been manipulated such that neither a functional p19INK4d protein nor a functional p27KIP1 protein is expressed in a defined culture environment, e.g., by using a specific inducible promoter element that can be repressed in culture. However, the functional proteins are expressed after implanting the cells into a subject animal upon de-repression of the promoter in vivo (e.g., the repressor is not present in vivo). In this manner neuronal cells, for example can be proliferated ex vivo, yet still function identically to the wild type cells i.e., express functional p19INK4d protein and functional p27KIP1 protein, when implanted into the subject. Alternatively, specific inhibitors of the expression and/or activity of the functional p19INK4d protein and functional p27KIP1 protein can be added to the culture medium and removed from the cells prior to implanting them into the subject.
In still another aspect of the present invention are methods of identifying a potential modulator of motor function and/or auditory function or vestibular function. In one such embodiment, the method uses a non-human knockout animal that can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein. In a preferred embodiment the non-human knockout animal is a mammal. In a particular embodiment of this type, the non-human mammalian knockout animal is a knockout mouse. A particular embodiment of this type comprises administering a potential modulator to the non-human knockout animal and measuring the response of the knockout animal to the potential modulator. The response of the knockout animal is then compared with that of an animal having a wild type p19INK4d gene and a wild type p27KIP1 gene. The potential modulator is selected based on the difference in response observed between the knockout animal in the presence and absence of the potential modulator relative to that of the animal having the wild type p19INK4d gene and the wild type p27KIP1 gene. In one such embodiment the life span of the animal is determined. An increase in life span of the knockout animal is indicative that the modulator is an antagonist for the effect due to the INK4d/KIP1 double null mouse. Similarly, the decrease in the magnitude of other characteristics of the INK4d/KIP1 double null mouse such as a reduction of seizures, and or magnitude of bradykinesia, or an increase in relative propriosensing are also indicative that the modulator may be useful for motor function disorders. Similarly, an increase in hearing and vestibular function, as measured by standard techniques such as the auditory brainstem response (ABR), would be indicative of the effectiveness of a specific modulator.
The present invention provides additional methods of selecting a modulator of neuronal cell and/or inner ear sensory epithelium proliferation. One such embodiment comprises administering a potential modulator to a neuronal cell that has been manipulated to be missing all or essentially all of an activity of the p19INK4d protein. The amount of proliferation of the neuronal cell is determined and a modulator is selected when the amount of proliferation determined in the presence of the potential modulator is greater than that determined in the absence of the potential modulator. A related embodiment comprises administering a potential modulator to a neuronal cell that has been manipulated to be missing all or essentially all of an activity of the p27KIP1 protein. The amount of proliferation of the neuronal cell is determined and a modulator is selected when the amount of proliferation determined in the presence of the potential modulator is greater than that determined in the absence of the potential modulator. In this manner inhibitors of both proteins can be individually identified. Such inhibitors can be combined in a pharmaceutical composition along with a physiological carrier to treat neurological degenerative diseases for example.
In still another embodiment the present invention provides methods of selecting a modulator of neuronal cell proliferation using a mammalian cell that can neither express a functional p19INK4d protein nor express a functional p27KIP1 protein. One such embodiment comprises administering a potential modulator to the neuronal cell and determining the amount of proliferation of the neuronal cell. A modulator of neuronal cell proliferation is selected when the amount of proliferation determined in the presence of the potential modulator is different from that determined in the absence of the potential modulator. A modulator is selected as an agonist when the amount of cellular proliferation increases whereas a modulator is selected as an antagonist when the amount of cellular proliferation decreases. Such an antagonist could be useful in preventing detrimental neural growth. All of the modulators identified by the methods of the present invention are also part of the present invention.
Accordingly, it is a principal object of the present invention to provide a non-human animal that is incapable of expressing functional p19INK4d and incapable of expressing functional p27KIP1 protein.
It is a further object of the present invention to provide a non-human animal that is capable of serving as an animal model for studying a motor disorders having symptoms that include bradykinesia and/or proprioceptive abnormalities and/or seizures.
It is a further object of the present invention to provide a mammalian cell that is capable of being used to identify agonists and antagonists of neuronal cell growth.
It is a further object of the present invention to provide methods of stimulating neuronal cell growth in cell culture.
It is a further object of the present invention to provide a method for stimulating growth of neuronal populations that are lost due to degenerative diseases, or due to traumatic injury in a subject animal.
It is a further object of the present invention to provide a method of treating subjects in need of neuronal cells by implanting into the subject a neuronal cell that has been manipulated to be incapable of expressing functional p19INK4d and incapable of expressing functional p27KIP1 protein, at least during the period that the cell is cultured ex vivo.
It is a further object of the present invention to provide a method of screening drug libraries for agents that can modulate neuronal cell growth.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.